It has long been recognized that the fluorescent and phosphorescent properties of certain materials vary in accordance with properties of the surroundings. For example, certain luminescent materials are subject to "quenching" or extinction of their luminescent response by oxygen. Quenching reduces the lifetime of the luminescence,, and also reduces the intensity of the luminescence. Other luminescent materials can be quenched by other chemical substances. Still others have luminescent properties which vary with temperature. In laboratory experiments where substantial amounts of luminescent materials can be directly illuminated and observed under readily controlled conditions, either the total luminescent intensity or the lifetime of the luminescence can be observed.
Chemical compositions of interest may have decay times ranging from seconds to picoseconds. With very short-lived luminescence, it is normally not practical to obtain useful information simply by exposing the composition to light, terminating the exposure and observing the decay of luminescence directly.
However, it is possible to obtain equivalent information by exposing the composition to excitation light having amplitude varying at a predetermined excitation modulation frequency and observing the luminescent response of the composition. Typically, the response includes a component varying in intensity at the same frequency as the excitation light. One characteristic of the emitted light which can be observed is the degree of modulation at the particular frequency used, i.e., the ratio between the intensity of the component at this frequency and the total intensity of the emitted light. Another characteristic of the emitted light which can be observed is the phase relationship between the cyclic variations in the emitted light and the cyclic variations in the excitation light. It has been the practice heretofore to conduct experiments of this nature at numerous modulation frequencies and gather information such as degree of modulation, phase angle and the like at each such frequency. Depending on the lifetime of the decay being studied, the modulation frequencies may be in the Hertz to gigahertz range. Utilizing known techniques, significant information concerning the physical and chemical characteristics of the composition can be deduced from the information gathered using plural frequencies. These techniques are commonly referred to as "frequency-domain fluorometry" and "frequency-domain phosphorimetry".
In laboratory experiments, such frequency domain techniques can distinguish between plural, simultaneously-occuring decays having different lifetimes. Thus, Bright et al, Rapid-Scanning Frequency Domain Fluorometer with Picosecond Time Resolution, Applied Optics, Vol. 26 Number 17,pp. 3526-3529, notes that a frequency domain fluorometer can resolve the three different decay times of three different reagents in admixture with one another. Lakowicz et al, A 2GHz Frequency-domain Fluorometer . . . , SPIE Vol 743, Fluorescence Detection (1987, pp 2-8) notes that a frequency domain fluorometer can resolve plural decay times associated with plural modes of decay of a single substance.
In frequency domain phosphorimetry and fluorometry, the various excitation modulation frequencies normally have been applied in sequence, one frequency at a time. Substantial time is required to collect data for all of the various frequencies. This approach is unsuitable for application in dynamic systems where the composition is changing with time. Accordingly, there have been attempts made to obtain similar information by applying light at various frequencies simultaneously and then measuring the response at all of these various frequencies simultaneously.
Mitchell et al, U.S. Pat. No. 4,939,457 discloses a laboratory instrument in which the excitation light is applied as series of pulses at a fundamental pulse-repetition frequency. This pulsatile excitation light includes components amplitude-modulated at the fundamental frequency and at harmonics or integral multiples of that frequency. The response or emitted light likewise includes components modulated at all of these frequencies. Pulsatile mixing signal having a fundamental frequency and harmonic frequencies slightly different from the corresponding frequencies in the excitation signal is also generated. The gain of the detector used to convert the response light into electrical signals is varied in accordance with the mixing signal, thereby mixing the response light signal with the mixing signal. The resulting cross-correlated or mixed signal has a fundamental frequency equal to the difference between the fundamental modulation frequency of the response light and the fundamental frequency of the mixing signals, and has harmonics at frequencies equal to the differences between corresponding harmonics of the response and mixing signals. In theory, the cross-correlated signal is a replica of response light, but with low modulation frequencies such that the cross-correlated signal can be digitized by conventional digital sampling devices. These digital samples of the cross-correlated signal versus time are then converted via Fourier transformation to a frequency-domain representation, including phase and modulation values for the various frequencies. This approach, however, encounters substantial degradation of signal strength at the higher harmonics, and hence is useful only with relatively strong emitted light signals.
Various instruments have been proposed to exploit luminescent phenomena in chemical and/or physical measuring instruments. For example, U.S. Pat. No. 4,810,655 discloses an instrument for determining oxygen concentration by applying excitation light to a fluorescent material and observing the time dependence of fluorescence decay, as by determining a ratio of luminescence intensity between two different periods of time after excitation. As the oxygen concentration in the environment surrounding the luminescent material changes, the pattern of fluorescent decay with time also changes. The '655 instrument employs a "light pipe" for transmitting the requisite excitation light to the luminescent material and for transmitting the light back to a sensor. European Patent Application 0,283,289 monitors the intensity of long lived phosphorescent emissions from a phosphorescent material bonded to an end of an optical fiber. The optical fiber is small enough that it can be inserted through a small tube, such as an intravenous catheter or the like, so that the phosphorescent material lies within a blood vessel and acts as an in vivo PO.sub.2 sensor. Other fiber optic based PP.sub.2 sensors are disclosed in U.S. Pat. No. 4,476,870 and European Patent Application 0,252,578.
Sensing of multiple phenomena with a single probe has also been suggested. Thus Kane, U.S. Pat. No. 4,758,814 describes a pH and PO.sub.2 sensor utilizing two fluorescent dyes which emit at different wavelengths. The emissions from one dye are affected by pH, whereas the emissions from another dye are affected by O.sub.2. Thus the emitted light at one wavelength provides a measure of pH, whereas the emitted light at another wavelength provides a measure of O.sub.2 concentration. Optical filtering is employed to segregate the response light from the two dyes so that their respective responses can be monitored separately.
Cubbers et al, U.S. Reissue Pat. No. 31,879 mentions the possibility of a composite luminescent element or "optode" having plural indicating substances embedded therein, again with signal segregation by light wavelength. Kelsius, Inc., PCT published application W088/05533 is directed generally to a sensor having a plurality of detectors incorporated therein.
These and other arrangements which rely on segregation by wavelength necessarily requires that the luminescent materials be selected to operate at different wavelengths. This restricts the choice of luminescent materials. Additionally, the required wavelength-restrictive optical filters may attenuate the desired wavelengths to some degreee, thus degrading the signal.
Although these and other fiber optic based luminescence probes and instruments have been proposed for monitoring chemical and/or physical conditions within the bodies of living subjects, the instruments available heretofore have suffered from certain significant drawbacks. For ease of insertion into the body through a needle or intravascular catheter, a fiber optic probe should be less than about 450 micrometers in diameter. The amount of luminescent material which can be accommodated in a probe of such small diameter is limited. The excitation light intensity must be maintained at a reasonable level to avoid destruction of the luminescent materials. For a given intensity level, the total excitation light energy which can be transmitted along the fiber optic is directly proportional to the cross-sectional area of the fiber optic. Thus, only limited light energy can be applied to excite the luminescent material. All of these factors tend to limit the amplitude of the response light emitted by the luminescent material and transmitted back along the fiber to the proximal end. Even highly sensitive photodetectors will provide only a weak signal. The signal from the actual luminescent material at the probe may be effectively hidden by the background noise. Stated another way, such instruments have had poor signal to noise ratios. This problem has been particularly severe in the case of instruments arranged to monitor the decay rate of relatively shortlived luminescent phenomena such as fluorescence or rapidly-decaying phosphorescence.
The frequency domain approach has not been generally applied in luminescence-based fiber optic sensing instruments. Instruments using one modulation frequency at a time acquire the necessary data too slowly for practical in vivo sensing. The signal attenuation encountered with the multiple harmonic cross-correlation approach normally renders this approach impractical for use with fiber optic systems.
The aforementioned U.S. Patent Application 07/481,406 discloses a substantial advance in frequency domain techniques. Instruments according to that application preferably include means for applying excitation light such as a series of pulses incorporating a plurality of excitation modulation frequency so that the luminescent material emits response light varying in amplitude at the same plural modulation frequencies. The excitation light typically is applied as a series of pulses at a fundamental with pulse frequency, so that the excitation light, and intensity response light emitted by the luminescent material include components varying at the fundamental and at harmonics of that frequency. One aspect of that disclosure involves the uses of direct sampling and, preferably, digitization, of the response light. For example, the response light may be converted to an electrical signal by a photodetector such that the instantaneous electrical signal from the photodetector is directly related to the instantaneous intensity or amplitude of the response light, and that signal may be directly sampled. The sampled response signal desirably is converted to a frequency domain representation of the response light, incorporating values of the modulation ratio and phase at each frequency. Preferably, a reference or excitation signal representing the excitation light is sampled, digitized and converted in substantially the same way so that the characteristics of each frequency in the response signal can be compared with the corresponding characteristics of the excitation light. For example, the phase of the response component at a particular modulation frequency can be compared directly with the phase of the excitation component at the same frequency. Preferred apparatus and methods according to the '406 application utilize sampling frequencies lower than the modulation frequencies. Various samples taken on different repetitions of the response and sample waveforms occur at different points along these waveforms and hence provide a complete sampling of the repetitive waveforms, in much the same way as if the sampling device operated at a much higher frequency. This arrangement permits effective sampling of very high frequency components, in the megahertz and gigahertz ranges using practical sampling devices. Because the response waveform (and reference waveform, where employed) are sampled directly without the need for mixing or cross-correlation prior to sampling, the signal loss in the higher harmonics associated with cross-correlation is effectively eliminated.
As described in greater detail in the aforementioned '131 application, such direct sampling and conversion techniques make it practical to apply frequency domain techniques to instruments using a fiber optic or similar light transmissive member and, particularly, a relatively small fiber optic probe as may be employed in monitoring chemical conditions within the body of a living subject. In particular, instruments using the technique of sampling and converting the response light signal to a frequency domain representation can be used with fiber optic probes insertable through an intravascular catheter insertable into the body of the subject through an intravascular catheter or similar device. Such instruments may employ luminescent materials having very fast decay times, and can detect very small changes in the decay times. This provides numerous benefits and generally superior performance to that achievable with other systems such as monitoring of emission intensity.